Article pubs.acs.org/Macromolecules
A Library of Thermoresponsive, Coacervate-Forming Biodegradable Polyesters John P. Swanson,† Leanna R. Monteleone,‡ Fadi Haso,† Philip J. Costanzo,‡ Tianbo Liu,† and Abraham Joy*,† †
Department of Polymer Science, The University of Akron, Akron, Ohio 44325, United States Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, California 93407, United States
‡
S Supporting Information *
ABSTRACT: We report on a new class of thermoresponsive biodegradable polyesters (TR-PE) inspired by polyacrylamides and elastin-like proteins (ELPs). The polyesters display reversible phase transition with tunable cloud point temperatures (Tcp) in aqueous solution as evidenced by UV−vis spectroscopy, 1H NMR, and DLS measurements. These polyesters form coacervate droplets above their lower critical solution temperature (LCST). The Tcp of the polyesters is influenced by the solutes such as urea, SDS, and NaCl. The Tcp of the copolymers shows a linear correlation with the composition of the polyesters indicating the ability to tune the phase change temperature. We also show that such thermoresponsive coacervates are capable of encapsulating small molecules such as Nile Red. Furthermore, the polyesters are hydrolytically degradable.
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INTRODUCTION Stimuli-responsive, or “smart”, materials have found wide acceptance across a variety of fields due to their ability to exhibit a significant change in physical properties as a result of a minor change in external stimuli such as light, electric potential, pH, redox, magnetic field, pressure, or temperature.1,2 Temperature-sensitive polymers, mainly represented by poly(Nisopropylacrylamide) (PNIPAM) 3,4 and polyoxazolines (POx),5,6 undergo a thermally induced reversible hydrophobicity change at a lower critical solution temperature (LCST). Above the LCST, certain thermoresponsive polymers such as PNIPAM undergo a coil−globule transition that results in significant dehydration of the polymer. The dehydrated polymers then aggregate due to hydrophobic interactions and the solution optically changes from clear to opaque. The temperature at which this observable macroscopic transformation occurs is defined as the cloud point temperature (Tcp) and is often used as a qualitative measure of the LCST.7 Not all temperature-responsive polymers undergo the coil− globule transition exhibited by PNIPAM. Instead, some polymers display an incomplete dehydration when brought above the LCST. The partially dehydrated polymers then separate into polymer-rich coacervate droplets within a polymer-deficient liquid phase. Similar to coil−globule transition-type polymers, coacervate-type polymers exhibit a thermally reversible cloud point in solution. Coacervate-type polymers are particularly attractive from a biomaterials standpoint since their incomplete dehydration leads to minimal conformational change as compared to coil−globule polymers. © 2015 American Chemical Society
This prevents coacervate-type polymers from damaging sensitive biomolecules and thus allows them to be used as agents for the purification of proteins and nucleic acid without disrupting their function,8 as controlled delivery agents for sensitive physiologically active molecules,9 and as injectable scaffolds.10,11 Despite this advantage, there have been far fewer reports in the literature on thermoresponsive coacervate-type polymers as compared to coil−globule type polymers. For example, certain elastin-like polypeptides (ELPs) display thermoresponsive coacervation and have been studied since the 1980s.12,13 Polymeric degradation is a desirable quality for numerous medical applications but is not achievable with many common thermoresponsive polymers, such as polyacrylamides.19 Polyamides such as ELPs and synthetic poly(amino acids) exhibit biodegradation due to the gradual hydrolysis of the amide backbone.20 However, the slow degradation may be limiting for applications where a faster degrading material, such as those based on a more hydrolyzable ester bond, would be desirable. Thermoresponsive polyphosphoesters21,22 and polyesters23−25 do exist in the literature but suffer from limited side chain functionality and few are able to form coacervates. Previously, our lab has synthesized a library of biodegradable “peptide-like” polyesters with a variety of bio-inspired pendant groups including orthogonal groups allowing for covalent Received: March 19, 2015 Revised: May 21, 2015 Published: June 12, 2015 3834
DOI: 10.1021/acs.macromol.5b00585 Macromolecules 2015, 48, 3834−3842
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Macromolecules attachment of biologically relevant ligands.26 Similar to polypeptides, these polyesters contain a significant amount of oxygen and nitrogen atoms in the polymer backbone. Copolyesters containing many different pendant groups can be synthesized, giving the “peptide-like” polyesters tunable physical properties. Despite the relatively hydrophilic backbone, the “peptide-like” polyesters do not display room temperature aqueous solubility or thermoresponsive behavior. The present work details our efforts toward designing a novel modular thermoresponsive polyester (TR-PE) system that exhibits thermoresponsive coacervate formation and biodegradation. Owing to the structural similarities between our previous “peptide-like” polyesters and PAMs, POx, and ELPs (Figure 1), we hypothesized that polyesters with specific
by various thermoresponsive polyacrylamides were synthesized. Despite the lower functional group density of the polyesters as compared to polyacrylamides, it was believed that the more hydrophilic pendant groups would impart temperature-sensitive solubility to the resultant TR-PEs as compared to the POx-like HEA polyesters. As the LCST of each polyacrylamide is affected by the hydrophobicity of the amide, it was hypothesized that different acrylamide mimics would display varying thermoresponsivity in a similar pattern. The desired amine was first reacted with ethyl succinyl chloride to produce the corresponding ethoxy amide in quantitative yields. A transamidation reaction in neat DEA afforded the pure HESA monomer after silica gel flash chromatography. HESA monomers were then polymerized using room temperature carbodiimide-mediated polyesterification. Owing to the similar solubilities of the monomers, polyesters, DPTS, and DIC urea byproducts in common solvents, purification by precipitation proved unsuccessful. Instead, pure TR-PEs were obtained by dialysis against MeOH at room temperature for 24 h and drying under reduced pressure (Figure 3). The resultant polyesters were characterized by NMR, which proved the removal of DPTS and urea byproducts. Adjusting the stoichiometry of diol to diacid in the polymerization resulted in a variety of molecular weight TR-PEs that were analyzed via GPC. Thermally Induced Phase Transitions. Aqueous solutions (10.0 mg/mL) of TR-PEs were prepared by equilibrating the polyester in DI water overnight at 4 °C. High molecular weight (∼55 kDa) TR-iPrPE, TR-DEPE, and TR-PyrPE were water-soluble at low temperatures and showed a rapid increase in turbidity upon being brought to room temperature (Figure 4). Cloudy solutions of these polyesters could be returned to their initial transparent state upon cooling. Despite their lower molecular weights (∼25 kDa), TR-CPPE and TR-nPrPE were not completely soluble in aqueous conditions at 0 °C. The thermoresponsivity of aqueous TR-PE solutions was probed using UV−vis and 1H NMR. To minimize molecular weight influences, TR-PE polyesters of similar high molecular weight were chosen for analysis. As shown in Figure 4, a clear reversible Tcp was observed for three TR-PEs of similar molecular weight: TR-iPrPE (7.8 °C), TR-DEPE (11.9 °C), and TR-PyrPE (15.8 °C). As hypothesized, the Tcp was modulated by amide side chain identity. However, the Tcp trend for TR-PEs did not seem to follow that for polyacrylamide (Figure 2), as TR-CPPE was expected to have a Tcp near that of TR-PyrPE but was instead insoluble. Similarly, TR-DEPE and TR-iPrPE were expected to have comparable Tcp but instead showed a 4.1 °C difference. These differences will be discussed in further detail below. Compared to thermoresponsive polyacrylamides, the TR-PEs exhibit a more significant hysteresis with TR-iPrPE being unable to fully rehydrate within the experimental time frame. A small increase in hysteresis is commonly observed when intra- and intermolecular hydrogen bonds form in the dehydrated state, making rehydration more difficult.32 Given the hydrogen bond donating secondary amide of TR-iPrPE and numerous hydrogen bond accepting oxygen and nitrogen atoms in the polyester, it is likely that such intra- or intermolecular hydrogen bonds are influencing the significant hysteresis as compared to TR-DEPE and TR-PyrPE. It has been reported that the selfassembly of complex structures such as β-turns can increase hysteresis by up to 20 °C in the case of certain ELPs.9
Figure 1. Chemical structure of polyacrylamides (PAM), polyoxazolines (POx), elastin-like polypeptides (ELPs), “peptide-like” polyesters, and thermoresponsive polyesters (TR-PE, this work).
pendant groups would exhibit thermoresponsive behavior. We designed a series of POx and PAM pendant group mimics in order to modulate the hydrophilicity and resulting Tcp. Similar to ELPs, we hypothesized that the multiple hydrogen bonding sites in the polyester backbone would prevent complete dehydration of the polyester leading to thermoresponsive coacervate formation. Finally, we predicted that ester bonds present in the polyester backbone would demonstrate similar degradation kinetics as observed in our previous “peptide-like” polyesters.
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RESULTS AND DISCUSSION Synthesis and Characterization of Monomer and Polyesters. It is well-known that the polyoxazolines PiPOx and PnPrOx exhibit LCST behavior at 36 and 24 °C, respectively.29,30 Given the structural similarities of POx to our “peptide-like” polyesters, we initially synthesized two hydroxyethyl amide (HEA) polyesters containing isopropyl and n-propyl functional groups using room temperature carbodiimide mediated polymerization as shown in Scheme 1.31 Previous work by our lab has shown that this method allows for the synthesis of high molecular weight polyesters with narrow PDI in high yield.26 However, these polyesters proved to be insoluble in aqueous solutions even after 16 h at 4 °C. As such, it was assumed that the hydrophobic/hydrophilic balance of the polyester was not optimal to elicit a temperaturesensitive response. In order to increase the hydrophilicity of the polyesters, a set of five hydroxyethyl succinamide (HESA) monomers inspired 3835
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Macromolecules Scheme 1. Synthetic Route for the Preparation of Polyestersa
a Reagents and conditions: (i) diethanolamine, neat, 80 °C, 16 h; (ii) succinic acid, DIC, DPTS, CH2Cl2, 0 °C to room temperature, 48 h; (iii) ethylsuccinyl chloride, Et3N, CH2Cl2, 0 °C to room temperature, 1 h.
Figure 2. Thermoresponsive polyesters as the inspiration for TR-PE library. Synthetic polymers, such as those based on hydrophilic monomers like hydroxybutyl vinyl ether (HOBVE),14 2-hydroxyisopropylacrylamide (HIPAM),15,16 2-carboxyisopropylacrylamide (CIPAM),17 and glycidyl methacrylate (GMA),18 have also been shown to form thermoresponsive coacervate droplets.
dehydration and a coacervate-type response.16 Changes in peak intensity and shape are also observed above the Tcp of TRiPrPE and TR-DEPE (see Supporting Information). It has been well established that the presence of an endothermic peak in a DSC measurement is one of the most accurate and robust methods for determining the LCST of thermoresponsive polymer solutions.33 The endothermic peak appears at the LCST when hydrogen bonds between structured water molecules and the polymer break, resulting in a large gain of entropy that compensates for the loss of entropy incurred by the dehydrated polymer.34 However, no endothermic peak was observed for any TR-PE solution even at high concentrations (150 mg/mL) despite showing a clear cloud point transition. This behavior is indicative of coacervate-type polymers, in which the partial dehydration of fewer structured waters leads
Preliminary attempts at determining if the TR-PEs exhibit any ordered structures were carried out by analysis of dilute TR-PE solutions. However, these studies did not show any distinguishing circular dichroism (CD) signals and the lack of any chiral center in the polyesters may preclude any ordered structures for these polyesters. Variable temperature 1H NMR in D2O was used to examine the change in polyester hydration with temperature. A representative example of TR-PyrPE is shown in Figure 5. Above the Tcp at 20 °C, the peaks corresponding to the DEA backbone begin to decrease in intensity and shift from two to three peaks as dehydration occurs. Moreover, the pyrrolidinyl alkyl peaks are seen to decrease in intensity and transform. Unlike coil−globule polymers, no peaks are observed to completely disappear, indicating partial instead of total 3836
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Figure 3. TR-PE library.
Figure 5. Variable temperature 1H NMR spectra of TR-PyrPE (Tcp = 15.8 °C) in D2O above and below cloud point.
monodisperse ELPs (Figure 6B).41 Increasing the concentration of hydrogen bond disrupting NaCl is well-known to promote thermoresponsive polymer collapse42,43 (“salting out” effect) and was observed to decrease the Tcp in a linear fashion (Figure 6C). Conversely, increasing the amount of SDS surfactant, which stabilizes the hydrated polyester, was observed to raise the Tcp (Figure 6D), in agreement with previous literature precedent.3,38,40 Furthermore, it was noted that higher concentrations of SDS were able to solubilize TR-CPPE and TR-nPrPE which allowed for a temperature response. PNIPAM has been previously used as a model to investigate the mechanism of protein denaturation by urea.44 Despite numerous investigations, this mechanism is still not well understood. The addition of urea results in a decrease of PNIPAM’s LCST, which is hypothesized to be the result of urea interacting bivalently with the polymer and stabilizing its dehydrated state via intra- and intermolecular hydrogen bonding.45,46 An opposite effect is observed for ELPs in which the LCST increases with urea concentration, as the hydrated state is made more stable. The current TR-PE system was observed to display a linear increase in Tcp with increasing urea content, similar to that of ELPs (Figure 6E). The resultant increase in thermoresponsive solubility was also seen for TRCPPE and TR-nPrPE. This behavior strongly indicates that despite the monomers being inspired by acrylamides, in solution TR-PEs appear to behave similar to ELPs. In the future, TR-PEs may provide a better model to understand the role of urea in protein denaturation. Tuning Phase Transition Temperature via Copolymerization. As TR-PE homopolyesters displayed distinctive Tcp, it was believed that thermoresponsivity could be tuned by copolymerization of different monomers, similar to other thermoresponsive polyacrylamides,15,33,34 polyesters,23 polyphosphoesters,21 and polyamides.20,47−49 It has been previously established that the hydrophobic/ hydrophilic balance can be used to control the behavior of thermoresponsive polymers. Within the realm of coacervationtype polymers, Sugihara et al. synthesized three types of poly(hydroxyethyl vinyl ether-co-isobutyl vinyl ether): random copolymers, diblock copolymers, and “block and random” copolymers.50 Of these, only the random copolymers exhibited
Figure 4. Reversible cloud point behavior of TR-PyrPE (top, Tcp = 15.8 °C, 10 mg/mL) and temperature-dependent transmittance of TRiPrPE, TR-DEPE, and TR-PyrPE (bottom, Mn ∼ 55 kDa, 10 mg/mL, 1 °C/min) in DI water exhibiting clear hysteresis.
to a reduced amount of entropy gained (relative to coil− globule polymers).15,16 The loss of entropy due to collapse of the polymer chain cannot be overcome by smaller entropy gained from partial dehydration of structured water molecules, leading to reduction or elimination of the endothermic peak. The absence of an endothermic peak in DSC strongly indicated that TR-PEs were coacervate-type polymers. Effect of Polyester Concentration, Molecular Weight, and Cosolutes on Phase Transition. In order to further explore the aqueous solution properties of the TR-PE system, UV−vis was used to probe the effect of polyester concentration, molecular weight, and cosolutes on Tcp. As expected, increasing polyester concentration increases the polyester available to form coacervates and results in an earlier onset of Tcp (Figure 6A) similar to what is seen for other thermoresponsive systems.11,38,40 Increasing the molecular weight of TR-PEs decreases their solubility, in turn logarithmically decreasing the Tcp. This behavior is similar to what is observed with more 3837
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Macromolecules Table 1. Characterization of TR-PEs polyester
Mna (kDa)
Mwa (kDa)
Đa
Tgb (°C)
Tcpc (°C)
Tcp PAMd (°C)
coacervate [polyester]e (%)
coacervate [polyester]e (mg/mL)
coacervate droplet Rhf (nm)
TR-CPPE TR-nPrPE TR-iPrPE TR-DEPE TR-PyrPE
25.5 29.1 56.5 56.5 54.2
42.4 43.9 88.5 87.6 86.6
1.6 1.5 1.6 1.5 1.5
13.8 4.8 10.0 −4.2 0.2
N/A N/A 7.8 11.9 15.8
5335 1036 3237 3338 5139
64.6 65.4 61.4 57.3 44.2
1830 1890 1590 1340 792
N/A N/A 102 118 226
a
Determined by DMF GPC relative to PMMA standards. bDetermined by DSC. cDefined as 50% transmission during temperature controlled UV− vis analysis. dCorresponding values of thermoresponsive polyacrylamides. eAverage of three measurements. fDetermined using temperature controlled DLS analysis.
Figure 6. Aqueous solution properties of TR-PEs (10 mg/mL unless otherwise stated) as a function of concentration (A), molecular weight (B), NaCl (C), and SDS (D), and urea (E).
feed,26 it was expected that the composition of TR-PEs could be reasonably controlled by the monomer feed as well, resulting in control over the Tcp. As shown in Figure 7, statistically random TR-PEs of similar high molecular weights display Tcp between that of their respective homopolyesters (Table 2), indicating that the temperature response can be tuned by monomer feed. Coacervate Analysis. Upon being left at room temperature overnight, the turbid aqueous solutions of TR-iPrPE, TRDEPE, and TR-PyrPE were observed to phase separate into a polymer-deficient aqueous phase and a viscous polymer-rich coacervate phase. As seen in Figure 9A, optical microscopy of the turbid solutions showed submicron-sized coacervate droplets. Centrifugation of the turbid solutions allowed for rapid coalescence of the coacervate droplets and analysis of the polyester concentration (Table 1). Following the trend witnessed in the Tcp experiments, polymer coacervate concentration did not seem to correlate with the hydrophobicity of the amide, which will be discussed in more detail below. The size of TR-PE coacervate droplets was quantified using DLS.
Table 2. Characterization of TR-PE Copolyesters monomer feed (iPr:Pyr)
polyester compositiona (iPr:Pyr)
Mnb (kDa)
Đb
CPc (°C)
100:0 75:25 50:50 25:75 0:100
100:0 71:29 48:52 27:73 0:100
56.6 52.2 56.0 55.0 54.2
1.6 1.4 1.4 1.4 1.5
7.2 7.9 9.5 13.1 15.8
a Determined by 1H NMR. bDetermined by DMF GPC relative to PMMA standards. cDefined as 50% transmission during temperature controlled UV−vis analysis.
a sharp phase separation above the Tcp, resulting in the formation of coacervate droplets. The authors suggested that the random sequence of the comonomers was crucial for welldefined phase separation and coacervate formation. Further more, Maeda et al. was able to show Tcp control of poly(NIPAM-ran-HIPAM), a thermoresponsive coacervatetype copolymer, by controlling the comonomer content.15,16 Given that the random “peptide-like” copolyester composition as determined by NMR was statistically similar to the monomer 3838
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Figure 8. CONTIN analysis of the DLS data of TR-DEPE (Tcp = 11.8 °C) above and below the cloud point.
Figure 7. Temperature-dependent transmittance of TR-PE copolyester solutions (Mn ∼ 55 kDa).
As shown in a representative CONTIN analysis of the DLS measurements of TR-DEPE (Figure 8), the onset of coacervate formation is observed above the Tcp. Coacervate droplets are shown to be relatively monodisperse and stable for at least 3 h, making them potentially useful for a number of biomaterials applications such as controlled delivery of therapeutics. It should be noted that there is small difference between the onset of coacervate formation, as obtained from DLS measurements, and Tcp. This is primarily due the qualitative nature of Tcp experiments, which measure macroscopic changes and are affected by conditions such as concentration and heating rate. The results of DLS analysis show that the higher the Tcp and coacervate water concentration, the greater the size of the resulting coacervate droplets (Table 1). Coacervates droplets of TR-iPrPE (Tcp = 7.8 °C) begin at 100 nm, while those of TRPyrPE (Tcp = 15.8 °C) show an initial size of 220 nm. Based on the Tcp and coacervate data, it seems that the type of amide present on the TR-PE side chain plays a greater effect on the solution properties than originally hypothesized. For all three secondary amide-based TR-PEs, the Tcp is much lower than would be expected if only hydrophilicity was taken into account. The tertiary amide based TR-PEs show better solubility, higher coacervate water content, and less hysteresis. This difference is likely due to the intra- and intermolecular hydrogen bonding available to the secondary amide side chain (a hydrogen bond donor and acceptor) and the numerous hydrogen bond accepting oxygen and nitrogen atoms per repeat unit as compared to the tertiary amide side chains (only a hydrogen bond acceptor). As previously discussed, such hydrogen bonding is known to increase the hysteresis of thermoresponsive polymers and makes the resolvation of the polymers less favorable. Our lab is currently studying other amide side chains in order to further investigate this hypothesis. Nile Red Separation. In order to show the feasibility of TR-PE coacervates for the thermally induced encapsulation of desirable compounds, Nile Red was used as a model compound. Nile Red is a hydrophobic dye with limited water solubility. A drop of 50 mM Nile Red in DMSO was added to a solution of TR-DEPE, which was quickly brought to room temperature. Upon coacervation and centrifugation, the purple dye-rich coacervate phase is easily observable (see Supporting Information). In contrast, no such behavior was observed when
Figure 9. Optical micrograph (0.5 wt %, 40× magnification) of TRDEPE (Mn = 55 kDa, Tcp = 11.9 °C) showing coacervate droplets at room temperature under bright-field filter (A) and after introduction of Nile Red under bright-field (B) and TRITC filters (C). Scale bar is the same for all images.
an aliquot of Nile Red was added to a blank solution. As seen in Figure 9, the addition of Nile Red caused only the Nile Red swollen coacervates to emit a red fluorescence, indicating successful incorporation of the dye. Our lab is currently studying the encapsulation efficiency and release kinetics of the TR-PE system for more complex hydrophilic model drugs and proteins. Degradation Behavior. The polyester backbone of the TR-PE system possesses inherent hydrolytic degradability. This is a significant advantage for numerous biomedical applications as compared to nondegradable thermoresponsive polymers such as PNIPAM or the enzymatically cleavable backbone of poly(amino acids) like ELPs.51 As a model study, the degradation of TR-DEPE was monitored at 37 °C, and the decrease in Mn is shown in Figure 10. Over a period of 7 days, TR-DEPE degraded from 72.7 to 26.7 kDa, a 63% Mn loss. This is comparable to our previously reported degradation of the more hydrophobic p(mAla) “peptide-like” polyester, which degraded from 63.3 to 41.4 kDa (a 35% Mn loss) over the same 7 day period, 26 as well as for the degradation of thermoresponsive poly(MEMO/ME2MO-alt-SA) polyesters.23 3839
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Dialysis tubing (regenerated cellulose, MWCO 3500 Da) was obtained from Thermo Fisher Scientific. Deionized water was used to prepare polyester solutions unless otherwise stated. Characterization. 1H and 13C NMR spectra in CDCl3 of the monomers and polyesters were recorded on either a Varian Mercury 300 or 500 MHz spectrometer. Chemical shifts were recorded in ppm (δ) relative to solvent signals. Variable temperature 1H NMR spectra in D2O were recorded on a Varian INOVA 400 MHz spectrometer with 15 min equilibrations at each temperature. Glass transition temperatures (Tg) of the polyesters were determined using a TA Q2000 DSC with a liquid N2 cooling unit and a heating/cooling rate of 10 °C/min. Polyester molecular weights were analyzed on a TOSOH EcoSec HLC-8320 GPC equipped with a refractive index detector (RI) and UV detector. Separation occurred over two PSS Gram Analytical GPC Columns in series using 25 mM LiBr in DMF as eluent at a flow rate of 0.8 mL/min. The column and detector temperatures were maintained at 50 °C. Molecular weights were obtained relative to PMMA standards using the RI signal. Synthesis of HEA Monomers. Synthesis of HEA monomers was conducted according to the previously published procedures.26 As a representative example, methyl butyrate (5.00 mL, 43.9 mmol, 1 equiv) and DEA (9.23 g, 87.8 mmol, 2 equiv) were added to a roundbottom flask equipped with a magnetic stir bar and heated at 80 °C overnight. After removing displaced MeOH under reduced pressure, the crude compound was analyzed via TLC (10% MeOH in DCM, ninhydrin). The product was observed at Rf ∼ 0.45 while unreacted DEA remained at Rf ∼ 0.05. The crude mixture was purified via silica gel flash chromatography (15% MeOH in CH2Cl2). The product was dried under reduced pressure to afford pure 1a (5.25 g, 30.0 mmol, 65%). The 1a monomer was characterized via NMR and IR. Synthesis of HESA Monomers. Synthesis of ethoxysuccinamide precursors was conducted according to modified literature procedures.26,28 As a representative example, pyrrolidine (3.0 mL, 36.5 mmol, 1 equiv) and Et3N (5.20 mL, 37.3 mmol, 1.02 equiv) were dissolved in dry CH2Cl2 (40.0 mL) into a round-bottom flask equipped with a magnetic stir bar. The reaction was cooled to 0 °C and purged with nitrogen for 15 min. Ethylsuccinyl chloride (5.20 mL, 36.5 mmol, 1 equiv) was added dropwise, and the reaction turned opaque white. The reaction was brought to room temperature and allowed to stir for 1 h under nitrogen. The solution was then added to DI water and extracted (3 × 40.0 mL of CH2Cl2). The product was dried over MgSO4, filtered, and concentrated under reduced pressure to afford pure 4a (6.98 g, 35.1 mmol, 96%). In a round-bottom flask equipped with a magnetic stir bar 4a (6.73 g, 33.8 mmol, 1 equiv) and DEA (7.10 g, 67.5 mmol, 2 equiv) were added and allowed to heat at 80 °C overnight while stirring. After removing the displaced EtOH under reduced pressure, the crude compound was analyzed via TLC (15% MeOH in DCM, ninhydrin). A small amount of unreacted ethoxy amide (Rf ∼ 0.56) was observed along with the desired compound (Rf ∼ 0.40). The crude mixture was purified via silica gel flash chromatography (10−20% MeOH in CH2Cl2). The product was dried under reduced pressure to afford pure HESA monomer 4b (3.42 g, 13.2 mmol, 40%). The HESA monomer was characterized via NMR and IR. Polymerization of Monomers. Homopolyesters and copolyesters of various monomers with succinic acid were prepared according to previously published methods.26 As a representative example, in a round-bottom flask equipped with a magnetic stir bar, 3b (1.25 g, 4.85 mmol, 1 equiv), succinic acid (573 mg, 4.85 mmol, 1 equiv), and DPTS (570 mg, 1.94 mmol, 0.4 equiv) were dissolved in dry CH2Cl2 (4.5 mL, 1.0 mL/100 mmol COOH) and purged with nitrogen for 15 min while stirring. This mixture was then briefly heated to homogenize the solution. The reaction was cooled to 0 °C, and DIC (2.28 mL, 14.6 mmol, 3 equiv) was added dropwise via syringe. The reaction was allowed to come to room temperature and stirred for 24−48 h under nitrogen. The HEA polyesters 1b and 2b were purified by precipitation into a stirred solution of cold MeOH. Since the HESA monomers, polyesters, DPTS, and DIC urea byproducts all displayed similar solubilities in common organic solvents, 4c was purified via dialysis against MeOH for 24 h with solvent changes at 3, 6, and 16 h. The
Figure 10. Hydrolytic degradation of TR-DEPE over a period of 7 days; n = 3.
Based on this evidence, it is reasonable to expect that that the four other TR-PEs will exhibit similar degradation trends. It is likely that the formation of a polymer-rich coacervate phase reduced the rate of ester bond hydrolysis, resulting in a slower degradation than would occur with a completely soluble polyester. Conclusion. In this work, we have developed a novel biodegradable polyester system that exhibits reversible temperature-dependent coacervation. The high molecular weight polyesters were synthesized using room temperature carbodiimide-mediated condensation polymerization. The temperature of coacervation, as well as coacervate droplet size and polymer concentration, was observed to be dependent on the amide side chain structure. Although the monomers were inspired by acrylamides, the increase in Tcp of the polyesters in the presence of urea indicate that their coacervation mechanism is likely similar to that ELPs. Further evidence for the similarity of TR-PEs to ELPs was provided by the lack of thermal signals in DSC experiments, indicating an incomplete dehydration of the polymer phase. The modular architecture of the system allows for tuning of the thermoresponsivity through copolymerization as well as the possible incorporation of monomers capable of covalent attachment of physiologically useful ligands. This platform represents a significant step forward within the field of thermoresponsive biomaterials and shows promise as a system for drug or protein delivery, protein purification, and injectable scaffolds. By this method it would be possible to deliver sensitive biomolecules by simple mixing with the soluble polymer phase and encapsulated by a temperature change. The biodegradable nature of the polyesters theoretically allows for periodic delivery of therapeutics by this methodology.
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EXPERIMENTAL SECTION
Materials. Succinic acid (99%), ethyl succinyl chloride (98%), isopropylamine (99%), pyrrolidine (99%), propylamine (98%), cyclopropylamine (99%), triethylamine (Et3N, 98%), diethanolamine (DEA, 99%), sodium chloride (NaCl, 99%), urea (98%), and sodium dodecyl sulfate (SDS, 98%) were purchased from Acros Organic and used as received. N,N′-Diisopropylcarbodiimide (DIC, 99%) was purchased from Oakwood Chemical and used as received. 4(Dimethylamino)pyridinium-4-toluenesulfonate (DPTS) was prepared according to literature methods.27 Reagent grade dichloromethane (CH2Cl2) was purchased from Thermo Fisher Scientific and dried by distilling over anhydrous CaH2. Reagent grade tetrahydrofuran (THF) and methanol (MeOH) were used as received from Thermo Fisher Scientific. Silica gel (40−63 μm, 230 × 400 mesh) for flash chromatography was purchased from Sorbent Technologies, Inc. 3840
DOI: 10.1021/acs.macromol.5b00585 Macromolecules 2015, 48, 3834−3842
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Macromolecules dialysis mixture was dried under vacuum to obtain pure 4c polyester as a white amorphous solid. The 4c polyester was characterized by GPC and NMR. Coacervate Analysis. Optical microscopy was carried out on an Olympus IX81 motorized inverted microscope using either bright-field or TRITC channel filters. To image the coacervate droplets, aqueous polyester solutions (5.0 mg/mL) were brought to room temperature for 5 min and immediately imaged on glass slides. To probe coacervation polyester concentration, aqueous polyester solutions (15 mg/mL) were prepared and left at 4 °C overnight. The solutions were then moved to a static 37 °C incubator for 2 h. Incubated samples were centrifuged at 3600 rpm for 5 min to achieve rapid phase separation. The polyester-dilute supernatant was then carefully removed with a glass pipet leaving the polyester-rich coacervate phase at the bottom of the vial. The resulting polyester complex was then weighed and lyophilized. The coacervate polyester concentration was determined using the ratio of the dried polyester to the weight of the coacervate as the average of three separate samples. Cloud Point Measurements. Turbidity measurements were carried out on a Shimadzu UV-1800 UV−vis spectrophotometer equipped with a Shimadzu S-1700 thermoelectric single cell holder in a 1 cm quartz cell. Deionized water was used as a reference. Polyester solutions (10.0 mg/mL unless otherwise noted) were prepared in DI water and left at 4 °C overnight to ensure complete dissolution and equilibration. Solutions were equilibrated at 0 or 5 °C until no change in transmittance was observed. Transmittance was recorded as a function of temperature at 1 °C/min at a fixed wavelength (350 nm). Tcp was defined as the temperature at which the transmittance was 50%. Light Scattering. Dynamic light scattering (DLS) measurements of the homopolyester aqueous solutions (0.5 mg/mL) were performed on a Brookhaven light scattering spectrometer (BI-200SM) equipped with a temperature-controlled solid state laser (λ = 532 nm). All DLS measurements were obtained at 90° scattering angle. An intensity− intensity time correlation function was measured by means of a multichannel digital correlator, which was then processed using the CONTIN method to obtain the average hydrodynamic radius of the particles in solution. The solutions were filtered through a 0.45 μm PVDF filter into glass vials and equilibrated for 30 min at each temperature prior to measurements. Degradation Analysis. Aqueous polyester solutions (10.0 mg/mL in DI water) were placed in a static 37 °C incubator and removed at various time points. The polyester-dilute supernatant was then carefully removed with a glass pipet leaving the polyester-rich coacervate phase at the bottom of the vial. The resulting coacervate was rinsed with DI water and lyophilized. The molecular weight of the dried coacervate was analyzed via GPC.
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well as Prof. Matthew Becker and Gina Policastro (Univ. of Akron, Polymer Science) for guidance with optical microscopy images.
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ASSOCIATED CONTENT
S Supporting Information *
Detailed synthetic protocols, characterization data of monomers and polymers, and solution transmittance plots. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b00585.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (A.J.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This material is based upon work supported by the NSF Graduate Research Fellowship Program 2012141051 to J.P.S. Part of this work was supported by an NSF CAREER grant to A.J. We thank Nicholas Johnson (Univ. of Akron, Chemistry) for assistance with variable temperature NMR experiments as 3841
DOI: 10.1021/acs.macromol.5b00585 Macromolecules 2015, 48, 3834−3842
Article
Macromolecules
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DOI: 10.1021/acs.macromol.5b00585 Macromolecules 2015, 48, 3834−3842
